Saponin purification

ABSTRACT

Saponin extracts containing at least 93% QS-21 main peak and 0.25-3% 2018 component by UV absorbance at 214 nm, methods for making said extracts, their use as vaccine adjuvants and related aspects.

TECHNICAL FIELD

The present application generally relates to saponin extracts, inparticular extracts of Quillaja saponaria Molina, methods for theirmanufacture and to associated aspects.

BACKGROUND

Adjuvants are included in vaccines to improve humoral and cellularimmune responses, particularly in the case of poorly immunogenic subunitvaccines. Similar to natural infections by pathogens, adjuvants rely onthe activation of the innate immune system to promote long-lastingadaptive immunity.

The Adjuvant System 01 (AS01) is a liposome-based adjuvant whichcontains two immunostimulants, 3-O-desacyl-4′-monophosphoryl lipid A(3D-MPL) and QS-21 (Garcon and Van Mechelen, 2011; Didierlaurent et al.,2017). 3D-MPL is a non-toxic derivative of the lipopolysaccharide fromSalmonella minnesota which is a TLR4 agonist) and QS-21 is a naturalsaponin extract from the bark of the South American tree Quillajasaponaria Molina (Kensil et al., 1991; Ragupathi et al., 2011). AS01 isincluded in the recently developed vaccines for malaria(RTS,S—Mosquirix®) and Herpes zoster (HZ/su—Shingrix®), and in multiplecandidate vaccines in development against pathogens such as humanimmunodeficiency virus and Mycobacterium tuberculosis.

AS01 injection results in rapid and transient activation of innateimmunity in animal models. Neutrophils and monocytes are rapidlyrecruited to the draining lymph node (dLN) upon immunization. Moreover,AS01 induces recruitment and activation of MHCII^(high) dendritic cells(DC), which are necessary for T cell activation (Didierlaurent A.M. etal., 2014). Some data are also available on the mechanism of action ofthe components of AS01. 3D-MPL signals via TLR4, stimulating NF-κBtranscriptional activity and cytokine production and directly activatesantigen-presenting cells (APCs) both in humans and in mice (De Becker etal., 2000; Ismaili et al., 2002; Martin et al., 2003; Mata-Haro et al.,2007). QS-21 promotes high antigen-specific antibody responses and CD8⁺T-cell responses in mice (Kensil and Kammer, 1998; Newman et al., 1992;Soltysik et al., 1995) and antigen-specific antibody responses in humans(Livingston et al., 1994). Because of its physical properties, it isthought that QS-21 might act as a danger signal in vivo (Lambrecht etal., 2009; Li et al., 2008). Although QS-21 has been shown to activateASC-NLRP3 inflammasome and subsequent IL-1β/IL-18 release (Marty-Roix,R. et al., 2016), the exact molecular pathways involved in the adjuvanteffect of saponins have yet to be clearly defined.

As with any component of a product which is approved as a humanmedicament, production of QS-21 requires the use of approvedmanufacturing processes and careful control of final composition toensure that it meets the required specification. Modification ofexisting processes requires costly and time consuming re-validation, yetdeviations from specification also result in waste. There is acontinuing need for robust methods for the manufacture of QS-21 and forQS-21 material of defined composition.

SUMMARY OF THE INVENTION

The present invention provides a saponin extract containing at least 93%QS-21 main peak and 0.25-3% 2018 component by UV absorbance at 214 nm.

Also provided is a saponin extract containing at least 93% by UVabsorbance at 214 nm triterpenoid glycosides having by negative ionelectrospray mass spectrometry m/z of 1855.9, 1987.9 or 2001.9, and0.25-3% by UV absorbance at 214 nm triterpenoid glycosides having m/z2017.9.

Additionally provided is a saponin extract containing at least 93%:

-   -   by UV absorbance at 214 nm.

Further, there is provided a method for the manufacture of a saponinextract comprising the steps of:

-   -   (i) selecting a crude aqueous extract of Quillaja saponaria        Molina having a suitable 2018 component composition;    -   (ii) purifying the extract by reverse phase chromatography using        a polystyrene resin; and    -   (iii) purifying the extract by reverse phase chromatography        using a phenyl resin.

There is provided the use of a saponin extract of the present inventionin the manufacture of a medicament.

Also provided are adjuvant compositions and vaccine compositionscomprising a saponin extract of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 HPLC chromatogram of a crude aqueous Quillaja saponaria Molinabark extract

FIG. 2 HPLC-UV chromatogram of a crude aqueous Quillaja saponaria Molinabark extract

FIG. 3 UPLC-UV chromatogram of a crude aqueous Quillaja saponaria Molinabark extract

FIG. 4 UPLC-UV chromatogram of a polystyrene purified saponin extract

FIG. 5 UPLC-UV/MS chromatogram of a purified Quillaja saponaria Molinasaponin extract

FIG. 6 UPLC-UV/MS chromatogram detail of a purified Quillaja saponariaMolina saponin extract

FIG. 7 Extracted mass chromatograms for 1988 and 2002 molecular weightions of a purified Quillaja saponaria Molina saponin extract

FIG. 8 Combined centroid spectrum of purified Quillaja saponaria Molinasaponin extract

FIG. 9 CD4 T-cell responses at day 21 in gE AS01 vaccinated mice

FIG. 10 Antibody responses at day 21 in gE AS01 vaccinated mice

FIG. 11 Antibody responses at day 28 in gE AS01 vaccinated mice

BRIEF DESCRIPTION OF THE SEQUENCES IDENTIFIERS

-   -   SEQ ID No. 1: RTS polypeptide sequence    -   SEQ ID No. 2: M. tuberculosis H37Rv strain Rv1196 polypeptide        sequence    -   SEQ ID No. 3: M. tuberculosis H37Rv strain Rv0125 polypeptide        sequence    -   SEQ ID No. 4: M72 fusion polypeptide sequence    -   SEQ ID No. 5: M72-2his fusion polypeptide sequence    -   SEQ ID No. 6: Varicella zoster virus truncated gE polypeptide        sequence    -   SEQ ID No. 7: Conformationally constrained RSV PreF antigen        polypeptide sequence    -   SEQ ID No. 8: HIV TV1 gp120 polypeptide sequence    -   SEQ ID No. 9: HIV 1086.C gp120 polypeptide sequence

DETAILED DESCRIPTION OF THE INVENTION

As mentioned previously, any component of a product which is authorisedas a human medicament requires the use of approved manufacturingprocesses and careful control of final composition to ensure that itmeets the required specification. Deviations from specification resultin waste. However, safety and efficacy investigation relies upon thetesting of defined compositions, therefore adaptation of componentspecifications introduces risk. Modification of existing processesrequires costly and time consuming re-validation.

The present inventors have found that crude aqueous extract of Quillajasaponaria Molina varies in composition, in particular with respect to acomponent referred to herein as the 2018 component, and that it isdifficult to separate excess 2018 component by applying existingapproved manufacturing processes. Consequently, the present inventionprovides methods for achieving a consistent purified extract by the useof a crude aqueous extract of a defined composition.

The present invention provides a saponin extract containing at least 93%QS-21 main peak and 0.25-3% 2018 component by UV absorbance at 214 nm,in particular, wherein the monoisotope of the most abundant species is1987.9 m/z. Suitably the saponin extract contains at least 98% QS-21group by UV absorbance at 214 nm. Typically the saponin extract contains1% or less of lyo impurity, especially 1% or less of the largest peakoutside the QS-21 group by UV absorbance at 214 nm.

Of particular interest are saponin extracts containing at least 98%QS-21 group, at least 93% QS-21 main peak, 0.25-3% 2018 component, 1% orless of largest peak outside the QS-21 group by UV absorbance at 214 nmand wherein the monoisotope of the most abundant species is 1987.9 m/z.

Also provided is a saponin extract containing at least 93% triterpenoidglycosides having m/z of 1855.9, 1987.9 or 2001.9, and 0.25-3%triterpenoid glycosides having m/z 2017.9 by UV absorbance at 214 nm, inparticular, wherein the monoisotope of the most abundant species is1987.9 m/z. Desirably the saponin extract contains at least 93%triterpenoid glycosides having m/z of 1855.9, 1987.9 or 2001.9,excluding B-isomer and lyo impurity, and 0.25-3% triterpenoid glycosideshaving m/z 2017.9 by UV absorbance at 214 nm, in particular, wherein themonoisotope of the most abundant species is 1987.9 m/z. Suitably thesaponin extract contains at least 98% triterpenoid glycosides having m/zof 1517.7, 1711.8, 1855.9, 1987.9, 2001.9, 2017.9 or 2118 by UVabsorbance at 214 nm. Desirably the saponin extract contains at least98% triterpenoid glycosides having m/z of 1517.7, 1711.8, 1855.9,1987.9, 2001.9, 2017.9 or 2118, excluding the lyo impurity, by UVabsorbance at 214 nm. Typically the saponin extract contains 1% or lessof lyo impurity by UV absorbance at 214 nm. Suitably the saponin extractcontains 1% or less of any other peak by UV absorbance at 214 nm.

Of particular interest are saponin extracts containing at least 98%triterpenoid glycosides having m/z of 1517.7, 1711.8, 1855.9, 1987.9,2001.9, 2017.9 or 2118, at least 93% triterpenoid glycosides having m/zof 1855.9, 1987.9 or 2001.9, 0.25-3% triterpenoid glycosides having m/z2017.9, 1% or less of any other peak by UV absorbance at 214 nm andwherein the monoisotope of the most abundant species is 1987.9 m/z,especially wherein the saponin extract contains at least 98%triterpenoid glycosides having m/z of 1517.7, 1711.8, 1855.9, 1987.9,2001.9, 2017.9 or 2118, excluding the lyo impurity, and at least 93%triterpenoid glycosides having m/z of 1855.9, 1987.9 or 2001.9 excludingB-isomer and lyo impurity, 0.25-3% triterpenoid glycosides having m/z2017.9, 1% or less of any other peak by UV absorbance at 214 nm andwherein the monoisotope of the most abundant species is 1987.9 m/z.

Additionally provided is a saponin extract containing at least 93%:

-   -   by UV absorbance at 214 nm, in particular, wherein the        monoisotope of the most abundant species is 1987.9 m/z. Suitably        the saponin extract contains at least 98%:

-   -    Suitably the saponin extract contains 98% of the aforementioned        components and the 2118 component. Typically the saponin extract        contains 1% or less:

-   -   especially 1% or less of any other peak by UV absorbance at 214        nm.

Of particular interest are saponin extracts containing at least 98%:

at least 93%:

0.25-3%:

1% or less of any other peak by UV absorbance at 214 nm and wherein themonoisotope of the most abundant species is 1987.9 m/z. Especially ofinterest are saponin extracts containing at least 98%:

or 2118 component,at least 93%:

0.25-3%:

1% or less of any other peak by UV absorbance at 214 nm and wherein themonoisotope of the most abundant species is 1987.9 m/z.

In certain embodiments the saponin extracts contain at least 65%, suchas at least 70%, 1988 component as determined by UV absorbance at 214 nmand by relative ion abundance. In certain embodiments the saponinextracts contain 85% or less, such as 80% or less, 1988 component asdetermined by UV absorbance at 214 nm and by relative ion abundance.

In certain embodiments the saponin extracts contain 30% or less, such as25% or less, 1856 component by UV absorbance at 214 nm and by relativeion abundance. In certain embodiments the saponin extracts contain atleast 5%, such as at least 10%, especially at least 15% 1856 componentby UV absorbance at 214 nm and by relative ion abundance.

In certain embodiments the saponin extracts contain 6% or less, such as4% or less, 2002 component by UV absorbance at 214 nm and by relativeion abundance. In certain embodiments the saponin extracts contain atleast 0.5%, such as at least 1%, 2002 component by UV absorbance at 214nm and by relative ion abundance.

In certain embodiments the saponin extracts contain at least 0.5%, suchas at least 1%, or at least 2% 2018 component by UV absorbance at 214 nmand by relative ion abundance.

In certain embodiments the saponin extracts contain at least 65%, suchas at least 70%:

by UV absorbance at 214 nm and by relative ion abundance.

In certain embodiments the saponin extracts contain 30% or less, such as25% or less:

by UV absorbance at 214 nm and by relative ion abundance. In certainembodiments the saponin extracts contain at least 5%, such as at least10%, especially at least 15%:

by UV absorbance at 24 nm and by relative ion abundance.In certain embodiments the saponin extracts contain 10% or less, such as5% or less:

by UV absorbance at 214 nm and by relative ion abundance. In certainembodiments the saponin extracts contain at least 0.5%, such as at least1%,

by ion abundance by UV absorbance at 214 nm and by relative ionabundance.

In certain embodiments the saponin extracts contain at least 0.5%, suchas at least 1%, or at least 2%,

by UV absorbance at 214 nm and by relative ion abundance.

Quil A is a saponin preparation isolated from the South American treeQuillaja saponaria Molina and was first described as having adjuvantactivity by Dalsgaard et al. in 1974 (“Saponin adjuvants”, Archiv. fürdie gesamte Virusforschung, Vol. 44, Springer Verlag, Berlin, p243-254).Purified fractions of Quil A have been isolated by HPLC which retainadjuvant activity without the toxicity associated with Quil A (see, forexample, EP03622789). Various fractions have been found to have adjuvantactivity, such as QS-7, QS-17, QS-18 and QS-21, although their toxicityvaries considerably.

By the term ‘saponin extract’ as used herein is meant an extract ofQuillaja saponaria Molina.

By the term ‘triterpenoid glycosides’ as used herein is meant an entityor entities having a triterpenoid core derivatised by sugars which areattached via glycosidic bonds.

Certain structures herein have been determined by MS/MS, limitations ofthe technique in differentiating certain branching, stereochemistry andisomeric sugar species (e.g. apiose and xylose) means that somestructures are putative and based on an assumed conserved core. Putativestructures should therefore be taken to mean the actual structure of thecomponent which has otherwise been identified, in the event the putativestructure is incorrect for any reason.

By the term ‘2018 component’ is meant the triterpenoid glycosidesidentified as ‘2018 Peak’ in FIG. 6 . Suitably the 2018 component in theUPLC-UV/MS methods described herein has a retention time ofapproximately 4.5 min, the primary component of the peak having amonoisotopic molecular weight of 2017.9. The 2018 component may also beidentified in the UPLC-UV methods described herein with a retention timeof approximately 5.8 min. The primary 2018 component has been identifiedas having the putative structure.

by MS/MS.

By the term ‘1988 component’ is meant the triterpenoid glycosidesidentified as part of the QS-21 main peak in FIG. 6 and having amonoisotopic molecular weight of 1987.9. Suitably the 1988 component inthe UPLC-UV/MS methods described herein has a retention time ofapproximately 4.4 min and a monoisotopic molecular weight of 1987.9. The1988 component may consist of QS-21A V1:

and QS-21A V2

By the term ‘1856 component’ is meant the triterpenoid glycosidesidentified as part of the QS-21 main peak in FIG. 6 and having amonoisotopic molecular weight of 1855.9. Suitably the 1856 component inthe UPLC-UV/MS methods described herein has a retention time ofapproximately 4.4 min and a monoisotopic molecular weight of 1855.9. The1856 component may consist of:

By the term ‘2002 component’ is meant the triterpenoid glycosidesidentified as part of the QS-21 main peak in FIG. 6 and having amonoisotopic molecular weight of 2001.9. Suitably the 2002 component inthe UPLC-UV/MS methods described herein has a retention time ofapproximately 4.4 min and a monoisotopic molecular weight of 2001.9. The2002 component has been identified as having the putative structure:

by MS/MS.

By the term ‘lyo impurity’ is meant the triterpenoid glycosidesidentified as ‘Lyophilization Peak’ in FIG. 6 . Suitably the lyoimpurity in the UPLC-UV/MS methods described herein has a retention timeof approximately 4.7 min and the primary component of the peak having amonoisotopic molecular weight of 1855.9. The primary lyo impurity hasbeen identified as having the putative structure:

by MS/MS.

By the term ‘B-isomer’ is meant the triterpenoid glycosides identifiedas ‘B-isomer’ in FIG. 6 . Suitably the B-isomer in the UPLC-UV/MSmethods described herein has a retention time of approximately 4.0 minand the primary component of the peak having a monoisotopic molecularweight of 1987.9. The primary B-isomer component has been identified ashaving the putative structure:

by MS/MS.

By the term ‘1518 component’ is meant the triterpenoid glycosidesidentified as ‘1518 Peak’ in FIG. 6 . Suitably the 1518 component in theUPLC-UV/MS methods described herein has a retention time ofapproximately 4.2 min and the primary component of the peak having amonoisotopic molecular weight of 1517.7. The primary 1518 component hasbeen identified as having the putative structure:

by MS/MS.

By the term ‘1712 component’ is meant the triterpenoid glycosidesidentified as ‘1712 Peak’ in FIG. 6 . Suitably the 1712 component in theUPLC-UV/MS methods described herein has a retention time ofapproximately 4.6 min and the primary component of the peak having amonoisotopic molecular weight of 1711.8. The primary 1712 component hasbeen identified as having the putative structure:

by MS/MS.

By the term ‘2118 component’ is meant the triterpenoid glycosidesidentified as ‘4.607’ in FIG. 6 . Suitably the 2118 component in theUPLC-UV/MS methods described herein has a retention time ofapproximately 4.6 and the primary component of the peak having amonoisotopic molecular weight of 2118.

By the term ‘QS-21 main peak’ is meant the triterpenoid glycosidesidentified as ‘QS-21’ in FIG. 6 . Suitably QS-21 main peak in theUPLC-UV/MS methods described herein has a retention time ofapproximately 4.4 min and molecular weight components of 1855.9, 1987.9and 2001.9 m/z. The QS-21 main peak may consist of QS-21A V1:

QS-21 A V2:

1856 component:

and2002 component:

By the term ‘QS-21 group’ is meant the triterpenoid glycosidesidentified from the B-isomer to the peak preceding the lyo impurity inthe UPLC-UV/MS methods described herein as having a retention time fromapproximately 4.0 min to approximately 4.7 min and having primarymonoisotopic molecular weights of 1517.7, 1711.8, 1855.9, 1987.9,2001.9, 2017.9 or 2118. The QS-21 group may consist of QS-21A V1:

QS-21 A V2:

1856 component:

2002 component:

2018 component:

B-isomer:

1518 component:

1712 component:

and

2118 component.

By the term ‘largest peak outside QS-21 group’ is meant the largest peakby UV, detectable in the UPLC-UV/MS methods described herein which isnot part of the QS-21 group.

By the term ‘Preceding peak’ is meant the peak immediately preceding theQS-21 main peak in the HPLC-UV methods described herein (see FIG. 2 ).

By the term ‘m/z’ is meant the mass to charge ratio of the monoisotopepeak. Unless otherwise specified, ‘m/z’ is determined by negative ionelectrospray mass spectrometry.

By the term ‘ion abundance’ is meant the amount of a specified m/zmeasured in the sample, or in a given peak as required by the context.The mass chromatogram for the specified m/z may be extracted from the MStotal ion chromatogram in the UPLC-UV/MS methods described herein. Themass chromatogram plots the signal intensity versus time. Ion abundanceis measured as the area of the integrated peak. The area for a specifiedm/z/area for a relative reference m/z=relative abundance.

By the term ‘UV absorbance at 214 nm’ is meant the area of an integratedpeak in the UV absorbance chromatogram. The (area for a specifiedpeak)/(area of all integrated peaks in the chromatogram)×100=percentagearea for the specified peak.

By the term ‘UV absorbance at 214 nm and relative ion abundance’ is meantan estimate for the percentage of a given m/z for co-eluting species.(Percentage area for given UV peak)×(relative ion abundance for m/z ofinterest in given peak)/(sum of all relative ion abundance for givenpeak)=percentage of m/z of interest in the given UV peak, assumesrelative ion abundance included for all coeluting species.

By the term ‘wherein the monoisotope of the most abundant species is1988 m/z’ is meant the monoisotope of the most abundant species, firstpeak in the isotopic group with highest response per m/z should be m/z1987.9. The most abundant species may be determined by creating acombined spectrum across the entire total ion chromatogram using theUPLC-UV/MS method (negative ion electrospray) as described herein.

By the term ‘dried’ is meant that substantially all solvent has beenremoved. A dried extract will typically contain less than 5% solvent w/w(such as less than 5% water w/w). Suitably the dried extract willcontain 100 ppm or less acetonitrile (w/w).

Further, there is provided a method for the manufacture of a saponinextract comprising the steps of:

-   -   (i) selecting a crude aqueous extract of Quillaja saponaria        Molina having a suitable 2018 component composition;    -   (ii) purifying the extract by reverse phase chromatography using        a polystyrene resin; and    -   (iii) purifying the extract by reverse phase chromatography        using a phenyl resin.

Desirably the process comprises the steps of:

-   -   (i) selecting a crude aqueous extract of Quillaja saponaria        Molina having a suitable 2018 component composition;    -   (ii) purifying the extract by polyvinylpyrollidone adsorption;    -   (iii) purifying the extract by diafiltration, ultrafiltration or        dialysis; (iv) purifying the extract by reverse phase        chromatography using a polystyrene resin; and    -   (v) purifying the extract by reverse phase chromatography using        a phenyl resin; wherein step (ii) and (iii) may optionally be in        reverse order or undertaken concurrently, though are typically        in the order shown.

The crude aqueous extract of Quillaja saponaria Molina is obtained byaqueous extraction (but need not be in aqueous form, e.g. it maysubsequently have been dried, subjected to solvent exchange orreconstituted into a different solvent). By the term ‘crude aqueousextract of Quillaja saponaria Molina having a suitable 2018 componentcomposition’ is meant an extract having a 2018 component to QS-21 mainpeak ratio of 0.075 or lower, in particular 0.064 or lower (asdetermined by UPLC-UV absorbance at 214 nm). Desirably the ratio of 2018component/QS-21 main peak is at least 0.005, such as at least 0.01 asmeasured by UV absorbance at 214 nm.

Suitably the Preceding peak to QS-21 main peak ratio is 0.45 or lower,in particular 0.4 or lower (as determined by HPLC-UV absorbance at 214nm). The Preceding peak to QS-21 main peak ratio may be 0.05 or higher,in particular 0.1 or higher (as determined by HPLC-UV absorbance at 214nm).

Typically the crude aqueous extract is a bark extract. Suitably theQS-21 main peak content in an aqueous solution of crude aqueous extractof Quillaja saponaria Molina is at least 1 g/L, such as at least 2 g/L,especially at least 2.5 g/L and in particular at least 2.8 g/L (e.g. asdetermined by UV absorbance relative to a control sample of knownconcentration).

Suitably the step of selecting a crude aqueous extract of Quillajasaponaria Molina having a suitable 2018 component composition, includestesting the composition to determine the 2018 component content.

The step of purifying the extract by polyvinylpyrollidone adsorptioninvolves treatment of the extract with polyvinylpyrollidone resin.Typically the extract is agitated with the polyvinylpyrollidone resin.The extract may subsequently be separated from the polyvinylpyrollidoneresin with adsorbed impurities by filtration. This step of the processgenerally removes polyphenolic impurities such as tannins.

The step of purifying the extract by diafiltration, ultrafiltration ordialysis, is suitably purification by diafiltration. typically usingtangential flow. An appropriate example of a membrane is a 30 kDacut-off. This step of the process generally removes salts, sugars andother low molecular weight materials.

The step of purifying the extract by reverse phase chromatography usinga polystyrene resin typically uses acetonitrile and water as solvent,usually acidified with a suitable acid such as acetic acid. An exampleof a suitable resin is Amberchrom XT20. Chromatography may be undertakenusing isocratic conditions, though is typically operated under a solventgradient (continuous, such as linear, or stepped), such as thoseprovided in the Examples. This step of the process generally removesnon-saponin material and enriches the desired saponins. Selectedfractions may be pooled to maximise yield of material matching therequired criteria (typically % QS-21≥18%, as determined by UV absorbancefollowing HPLC-UV and 2018/QS-21 Ratio≤0.054, as determined by UVabsorbance following UPLC-UV). Each polystyrene chromatography run istypically at a scale of between 25-200 g of QS-21, such as between50-150 g and in particular between 70-110 g (amounts being based onQS-21 main peak content in the material by UV).

Purifying the extract by reverse phase chromatography using a phenylresin typically uses acetonitrile and water as solvent, usuallyacidified with a suitable acid such as acetic acid. Chromatography maybe undertaken using a solvent gradient (continuous, such as linear, orstepped), though is typically operated under isocratic conditions. Thisstep of the process provides the final purification of the desiredsaponins. Selected fractions may be pooled to maximise yield of materialmatching the required criteria (typically % QS-21 group ≥98.5, % QS-21main peak ≥94.5, 2002/1988 ≤0.027, %2018 ≤2.7%, main peak outside of theQS-21 group ≤1%, as determined by UPLC-UV/MS). Each phenylchromatography run is typically at a scale of between 4-40 g of QS-21,such as between 10-30 g and in particular between 13-21 g (amounts beingbased on QS-21 main peak content in the material by UV).

The method may comprise the further step of removing solvent to providea dried saponin extract. Consequently, the invention provides a methodfor the manufacture of a saponin extract comprising the steps of:

-   -   (i) selecting a crude aqueous extract of Quillaja saponaria        Molina having a suitable 2018 component composition;    -   (ii) purifying the extract by reverse phase chromatography using        a polystyrene resin;    -   (iii) purifying the extract by reverse phase chromatography        using a phenyl resin; and    -   (iv) removing solvent to provide a dried saponin extract.        The invention also provides a method for the manufacture of a        saponin extract comprising the steps of:    -   (i) selecting a crude aqueous extract of Quillaja saponaria        Molina having a suitable 2018 component composition;    -   (ii) purifying the extract by polyvinylpyrollidone adsorption;    -   (iii) purifying the extract by diafiltration, ultrafiltration or        dialysis;    -   (iv) purifying the extract by reverse phase chromatography using        a polystyrene resin;    -   (v) purifying the extract by reverse phase chromatography using        a phenyl resin; and    -   (vi) removing solvent to provide a dried saponin extract.        wherein step (ii) and (iii) may optionally be in reverse order        or undertaken concurrently, though are typically in the order        shown.

In order to improve drying efficiency, it may be desirable to undertakefurther steps of concentrating the extract, such as by capture andrelease using an appropriate technique, for example reverse phasechromatography (e.g. using a C8 resin), and/or exchanging the solvent inadvance of the drying step.

Also provided is a method for the manufacture of a saponin extractcomprising the steps:

-   -   (i) selecting a crude aqueous extract of Quillaja saponaria        Molina having a suitable 2018 component composition;    -   (ii) purifying the extract by polyvinylpyrollidone adsorption;    -   (iii) purifying the extract by diafiltration, ultrafiltration or        dialysis;    -   (iv) purifying the extract by reverse phase chromatography using        a polystyrene resin;    -   (v) purifying the extract by reverse phase chromatography using        a phenyl resin;    -   (vi) optionally concentrating the extract;    -   (vii) optionally exchanging the solvent; and    -   (viii) removing the remaining solvent to provide a dried saponin        extract;        wherein steps (vi) and (vii) may be optionally be in reverse        order or undertaken concurrently, though are typically in the        order shown.

Also provided is a method for the manufacture of a saponin extractcomprising the steps:

-   -   (i) selecting a crude aqueous extract of Quillaja saponaria        Molina having a suitable 2018 component composition;    -   (ii) purifying the extract by polyvinylpyrollidone adsorption;    -   (iii) purifying the extract by diafiltration, ultrafiltration or        dialysis;    -   (iv) purifying the extract by reverse phase chromatography using        a polystyrene resin;    -   (v) purifying the extract by reverse phase chromatography using        a phenyl resin;    -   (vi) concentrating the extract by reverse phase chromatography        using a C8 resin;    -   (vii) exchanging the solvent; and    -   (viii) removing the remaining solvent to provide a dried saponin        extract.

Concentration of the extract may be performed using any suitabletechnique. For example, concentration may be performed using a captureand release methodology, such as reverse phase chromatography, inparticular using a C8 resin. The reverse phase chromatography typicallyuses acetonitrile and water as solvent, usually acidified with asuitable acid such as acetic acid. Chromatography is typically operatedunder a solvent gradient, with the saponin extract captured in loworganic solvent and eluted in high organic solvent, in particular astepped solvent gradient.

Exchanging the solvent may be performed using any suitable technique, inparticular diafiltration, ultrafiltration or dialysis, especiallydiafiltration. Solvent exchange may be useful, for example, in reducingthe acetonitrile content such as described in WO2014016374. A suitablemembrane may be selected to allow solvent exchange while retaining thesaponin extract, such as a 1 kDa membrane.

Drying, by removing the solvent, may be undertaken by any suitablemeans, in particular by lyophilisation. During drying, degradation ofthe saponin extract can occur, leading to the formation of lyo impurity.Consequently, it is desirable to dry under conditions which limitformation of lyo impurity, such as by limiting the drying temperatureand/or drying time. Suitably removal of solvent is undertaken by asingle lyophilisation process. The extent of drying required will dependon the nature of the solvent, for example non-pharmaceuticallyacceptable solvents will desirably be removed to a high degree, whereassome pharmaceutically acceptable solvents (such as water) may be removedto a lesser degree.

Suitably the methods of the present invention are undertaken at a scaleof between 25-1000 g of QS-21, such as between 50-500 g and inparticular between 100-500 g (amounts being based on QS-21 main peakcontent in the material by UV).

Also provided is a method for identifying a crude aqueous extract ofQuillaja saponaria Molina for use in the manufacture of a purifiedsaponin extract, such as the saponin extracts of the invention, saidmethod comprising the steps of:

-   -   (i) determining the 2018 component to QS-21 main peak ratio by        UPLC-UV absorbance at 214 nm;    -   (ii) selecting a crude extract having a 2018 component to QS-21        main peak ratio of 0.075 or lower.

In one embodiment, the crude aqueous extract selected in step (ii) has a2018 component to QS-21 main peak ratio of 0.064 or lower.

The invention also provides a method for the determining the 2018component to QS-21 main peak ratio in a crude aqueous extract ofQuillaja saponaria Molina, said method comprising the steps of:

-   -   (i) determining the 2018 component content in the crude aqueous        extract of Quillaja saponaria Molina by UPLC-UV absorbance at        214 nm;    -   (ii) determining the QS-21 main peak content in the crude        aqueous extract of Quillaja saponaria Molina by UPLC-UV        absorbance at 214 nm; and    -   (iii) comparing the 2018 component content to the QS-21 main        peak content to determine the 2018 component to QS-21 main peak        ratio.

There is provided the use of a saponin extract of the present inventionin the manufacture of a medicament. Additionally provided is a saponinextract of the present invention for use as a medicament, in particularas an adjuvant. Also provided is an adjuvant composition comprising asaponin extract of the present invention.

The saponin extracts of the present invention may be combined withfurther adjuvants, such as a TLR4 agonist, in particularlipopolysaccharide TLR4 agonists, such as lipid A derivatives,especially a monophosphoryl lipid A e.g. 3-de-O-acylated monophosphoryllipid A (3D-MPL). monophosphoryl lipid A (3D-MPL). 3D-MPL is sold underthe name ‘MPL’ by GlaxoSmithKline Biologicals N.A. and is referredthroughout the document as 3D-MPL. See, for example, U.S. Pat. Nos.4,436,727; 4,877,611; 4,866,034 and 4,912,094. 3D-MPL can be producedaccording to the methods described in GB 2 220 211 A. Chemically it is amixture of 3-deacylated monophosphoryl lipid A with 4, 5 or 6 acylatedchains.

Other TLR4 agonists which may be of use in the present invention includeGlucopyranosyl Lipid Adjuvant (GLA) such as described in WO2008/153541or WO2009/143457 or the literature articles Coler RN et al. (2011)Development and Characterization of Synthetic Glucopyranosyl LipidAdjuvant System as a Vaccine Adjuvant. PLoS ONE 6(1): e16333.doi:10.1371/journal.pone.0016333 and Arias MA et al. (2012)Glucopyranosyl Lipid Adjuvant (GLA), a Synthetic TLR4 Agonist, PromotesPotent Systemic and Mucosal Responses to Intranasal Immunization withHIVgp140. PLoS ONE 7(7): e41144. doi:10.1371/journal.pone.0041144.WO2008/153541 or WO2009/143457 are incorporated herein by reference forthe purpose of defining TLR4 agonists which may be of use in the presentinvention.

A typical adult human dose of adjuvant will comprise a saponin extractat amounts between 1 and 100 ug per human dose. The saponin extract maybe used at a level of about 50 ug. Examples of suitable ranges are 40-60ug, suitably 45-55 ug or 49-51 ug, such as 50 ug. In a furtherembodiment, the human dose comprises saponin extract at a level of about25 ug. Examples of lower ranges include 20-30 ug, suitably 22-28 ug or24-26 ug, such as 25 ug. Human doses intended for children may bereduced compared to those intended for an adult (e.g. reduction by 50%).

The TLR4 agonists, such as a lipopolysaccharide, such as 3D-MPL, can beused at amounts between 1 and 100 ug per human dose. 3D-MPL may be usedat a level of about 50 ug. Examples of suitable ranges are 40-60 ug,suitably 45-55 ug or 49-51 ug, such as 50 ug. In a further embodiment,the human dose comprises 3D-MPL at a level of about 25 ug. Examples oflower ranges include 20-30 ug, suitably 22-28 ug or 24-26 ug, such as 25ug. Human doses intended for children may be reduced compared to thoseintended for an adult (e.g. reduction by 50%).

When both a TLR4 agonist and a saponin extract are present in theadjuvant, then the weight ratio of TLR4 agonist to saponin is suitablybetween 1:5 to 5:1, suitably 1:1. For example, where 3D-MPL is presentat an amount of 50 ug or 25 ug, then suitably QS-21 may also be presentat an amount of 50 ug or 25 ug per human dose.

Adjuvants may also comprise a suitable carrier, such as an emulsion(e.g. and oil in water emulsion) or liposomes.

Liposomes

The term ‘liposome’ is well known in the art and defines a generalcategory of vesicles which comprise one or more lipid bilayerssurrounding an aqueous space. Liposomes thus consist of one or morelipid and/or phospholipid bilayers and can contain other molecules, suchas proteins or carbohydrates, in their structure. Because both lipid andaqueous phases are present, liposomes can encapsulate or entrapwater-soluble material, lipid-soluble material, and/or amphiphiliccompounds.

Liposome size may vary from 30 nm to several um depending on thephospholipid composition and the method used for their preparation.

The liposomes of use in the present invention suitably contain DOPC, or,consist essentially of DOPC and sterol (with saponin and optionally TLR4agonist).

In the present invention, the liposome size will be in the range of 50nm to 200 nm, especially 60 nm to 180 nm, such as 70-165 nm. Optimally,the liposomes should be stable and have a diameter of ˜100 nm to allowconvenient sterilization by filtration.

Structural integrity of the liposomes may be assessed by methods such asdynamic light scattering (DLS) measuring the size (Z-average diameter,Zav) and polydispersity of the liposomes, or, by electron microscopy foranalysis of the structure of the liposomes. In one embodiment theaverage particle size is between 95 and 120 nm, and/or, thepolydispersity (Pdl) index is not more than 0.3 (such as not more than0.2).

Further Excipients

In a further embodiment, a buffer is added to the composition. The pH ofa liquid preparation is adjusted in view of the components of thecomposition and necessary suitability for administration to the subject.Suitably, the pH of a liquid mixture is at least 4, at least 5, at least5.5, at least 5.8, at least 6. The pH of the liquid mixture may be lessthan 9, less than 8, less than 7.5 or less than 7. In other embodiments,pH of the liquid mixture is between 4 and 9, between 5 and 8, such asbetween 5.5 and 8. Consequently, the pH will suitably be between 6-9,such as 6.5-8.5. In a particularly preferred embodiment the pH isbetween 5.8 and 6.4.

An appropriate buffer may be selected from acetate, citrate, histidine,maleate, phosphate, succinate, tartrate and TRIS. In one embodiment, thebuffer is a phosphate buffer such as Na/Na₂PO₄, Na/K₂PO₄ or K/K₂PO₄.

The buffer can be present in the liquid mixture in an amount of at least6 mM, at least 10 mM or at least 40 mM. The buffer can be present in theliquid mixture in an amount of less than 100 mM, less than 60 mM or lessthan 40 mM.

It is well known that for parenteral administration solutions shouldhave a pharmaceutically acceptable osmolality to avoid cell distortionor lysis. A pharmaceutically acceptable osmolality will generally meanthat solutions will have an osmolality which is approximately isotonicor mildly hypertonic. Suitably the compositions (when reconstituted, ifpresented in dried form) will have an osmolality in the range of 250 to750 mOsm/kg, for example, the osmolality may be in the range of 250 to550 mOsm/kg, such as in the range of 280 to 500 mOsm/kg. In aparticularly preferred embodiment the osmolality may be in the range of280 to 310 mOsm/kg. Osmolality may be measured according to techniquesknown in the art, such as by the use of a commercially availableosmometer, for example the Advanced® Model 2020 available from AdvancedInstruments Inc. (USA).

An “isotonicity agent” is a compound that is physiologically toleratedand imparts a suitable tonicity to a formulation to prevent the net flowof water across cell membranes that are in contact with the formulation.In some embodiments, the isotonicity agent used for the composition is asalt (or mixtures of salts), conveniently the salt is sodium chloride,suitably at a concentration of approximately 150 nM. In otherembodiments, however, the composition comprises a non-ionic isotonicityagent and the concentration of sodium chloride in the composition isless than 100 mM, such as less than 80 mM, e.g. less than 50 mM, such asless 40 mM, less than 30 mM and especially less than 20 mM. The ionicstrength in the composition may be less than 100 mM, such as less than80 mM, e.g. less than 50 mM, such as less 40 mM or less than 30 mM.

In a particular embodiment, the non-ionic isotonicity agent is a polyol,such as sucrose and/or sorbitol. The concentration of sorbitol may e.g.between about 3% and about 15% (w/v), such as between about 4% and about10% (w/v). Adjuvants comprising an immunologically active saponinfraction and a TLR4 agonist wherein the isotonicity agent is salt or apolyol have been described in WO2012/080369.

Suitably, a human dose volume of between 0.05 ml and 1 ml, such asbetween 0.1 and 0.5 ml, in particular a dose volume of about 0.5 ml, or0.7 ml. The volumes of the compositions used may depend on the deliveryroute and location, with smaller doses being given by the intradermalroute. A unit dose container may contain an overage to allow for propermanipulation of materials during administration of the unit dose.

The ratio of saponin:DOPC will typically be in the order of 1:50 to 1:10(w/w), suitably between 1:25 to 1:15 (w/w), and preferably 1:22 to 1:18(w/w), such as 1:20 (w/w).

Suitably the saponin is presented in a less reactogenic compositionwhere it is quenched with an exogenous sterol, such as cholesterol.Cholesterol is disclosed in the Merck Index, 13th Edn., page 381, as anaturally occurring sterol found in animal fat. Cholesterol has theformula (C₂₇H₄₆O) and is also known as (3β)-cholest-5-en-3-ol.

The ratio of saponin:sterol will typically be in the order of 1:100 to1:1 (w/w), suitably between 1:10 to 1:1 (w/w), and preferably 1:5 to 1:1(w/w). Suitably excess sterol is present, the ratio of saponin:sterolbeing at least 1:2 (w/w). In one embodiment, the ratio of saponin:sterolis 1:5 (w/w). In one embodiment, the sterol is cholesterol.

The amount of liposome (weight of lipid and sterol) will typically be inthe range of 0.1 mg to 10 mg per human dose of a composition, inparticular 0.5 mg to 2 mg per human dose of a composition.

In a particularly suitable embodiment, liposomes used in the inventioncomprise DOPC and a sterol, in particular cholesterol. Thus, in aparticular embodiment, a composition used in the invention comprisessaponin extract in the form of a liposome, wherein said liposomecomprises DOPC and a sterol, in particular cholesterol.

Antigens

The adjuvants prepared according to the present invention may beutilised in conjunction with an immunogen or antigen. In someembodiments a polynucleotide encoding the immunogen or antigen isprovided.

The adjuvant may be administered separately from an immunogen or antigenmay be combined, either during manufacturing or extemporaneously, withan immunogen or antigen as an immunogenic composition for combinedadministration.

Consequently, there is provided a method for the preparation of animmunogenic composition comprising an immunogen or antigen, or apolynucleotide encoding the immunogen or antigen, said method comprisingthe steps of:

-   -   (i) preparing an adjuvant composition comprising a saponin        extract of the present invention;    -   (ii) mixing the adjuvant with an immunogen or antigen, or a        polynucleotide encoding the immunogen or antigen.

There is also provided the use of an adjuvant comprising a saponinextract of the present invention in the manufacture of a medicament.Suitably the medicament comprises an immunogen or antigen, or apolynucleotide encoding the immunogen or antigen.

Further provided is an adjuvant comprising a saponin extract of thepresent invention for use as a medicament. Suitably the medicamentcomprises an immunogen or antigen, or a polynucleotide encoding theimmunogen or antigen.

By the term immunogen is meant a polypeptide which is capable ofeliciting an immune response. Suitably the immunogen is an antigen whichcomprises at least one B or T cell epitope. The elicited immune responsemay be an antigen specific B cell response, which produces neutralizingantibodies. The elicited immune response may be an antigen specific Tcell response, which may be a systemic and/or a local response. Theantigen specific T cell response may comprise a CD4+ T cell response,such as a response involving CD4+ T cells expressing a plurality ofcytokines, e.g. IFNgamma, TNFalpha and/or IL2. Alternatively, oradditionally, the antigen specific T cell response comprises a CD8+ Tcell response, such as a response involving CD8+ T cells expressing aplurality of cytokines, e.g., IFNgamma, TNFalpha and/or IL2.

The antigen may be derived (such as obtained from) from a human ornon-human pathogen including, e.g., bacteria, fungi, parasiticmicroorganisms or multicellular parasites which infect human andnon-human vertebrates, or from a cancer cell or tumor cell.

In one embodiment the antigen is a recombinant protein, such as arecombinant prokaryotic protein.

In one embodiment, the antigen is derived from Plasmodium spp. (such asPlasmodium falciparum), Mycobacterium spp. (such as Mycobacteriumtuberculosis (TB)), Varicella Zoster Virus (VZV), human respiratorysyncytial virus, Human Immunodeficiency Virus (HIV), Moraxella spp.(such as Moraxella catarrhalis) or nontypable Haemophilus influenzae(ntHi).

The antigen may comprise or consist of preparations derived fromparasites that cause malaria such as Plasmodium falciparum or Plasmodiumvivax.

In one embodiment, the antigen may be the Plasmodium falciparumcircumsporozoite (CS) protein or a variant thereof. A suitable variantof the CS protein may be a variant wherein parts of the CS protein arein the form of a hybrid protein with the surface antigen S fromhepatitis B (HBsAg). The CS variant antigen may e.g. be in the form of ahybrid protein comprising substantially all the C-terminal portion ofthe CS protein, four or more tandem repeats of the CS proteinimmunodominant region, and HBsAg. The hybrid protein may comprise asequence which contains at least 160 amino acids and which issubstantially homologous to the C-terminal portion of the CS protein,but devoid of the hydrophobic anchor sequence. The CS protein may bedevoid of the last 12 amino-acids from the C terminal. Further, it maycontain 4 or more e.g. 10 or more Asn-Ala-Asn-Pro tetrapeptide (NANP)repeat motifs.

The hybrid protein for use in the invention may be a protein whichcomprises a portion of the CS protein of P. falciparum substantially ascorresponding to amino acids 207-395 of P. falciparum clone 3D7, derivedfrom the strain NF54 fused in frame via a linear linker to theN-terminus of HBsAg. The linker may comprise a portion of preS2 fromHBsAg. CS constructs suitable for use in the present invention areoutlined in WO93/10152, which granted in the US as U.S. Pat. Nos.5,928,902 and 6,169,171, both of which are incorporated by reference forthe purpose of describing suitable proteins for use in the presentinvention.

A particular hybrid protein for use in the invention is the hybridprotein known as RTS (SEQ ID No. 1, also described in WO2015/150568,WO93/10152 (wherein it is denoted RTS*) and in WO98/05355, whichconsists of:

-   -   a methionine residue    -   three amino acid residues, Met Ala Pro    -   a stretch of 189 amino acids representing amino acids 207 to 395        of the CS protein of P. falciparum strain 3D7    -   an glycine residue    -   four amino acid residues, Pro Val Thr Asn, representing the four        carboxy terminal residues of the hepatitis B virus (adw        serotype) preS2 protein, and    -   a stretch of 226 amino acids, encoded by nucleotides 1653 to        2330, and specifying the S protein of hepatitis B virus (adw        serotype).        RTS may be in the form of RTS,S mixed particles. RTS,S particles        comprise two polypeptides, RTS and S, that may be synthesized        simultaneously and spontaneously form composite particulate        structures (RTS,S).

The antigen may comprise or consist of preparations derived fromMycobacterium spp., such as Mycobacterium bovis or Mycobacteriumtuberculosis, in particular Mycobacterium tuberculosis.

Antigens of interest in the field of tuberculosis include Rv1196 andRv0125. Rv1196 (described, for example, by the name Mtb39a in Dillon etal Infection and Immunity 1999 67(6): 2941-2950) is highly conserved,with 100% sequence identity across H37Rv, C, Haarlem, CDC1551,94-M4241A, 98-R604INH-RIF-EM, KZN605, KZN1435, KZN4207, KZNR506 strains,the F11 strain having a single point mutation Q30K (most other clinicalisolates have in excess of 90% identity to H37Rv). Rv0125 (described,for example, by the name Mtb32a in Skeiky et al Infection and Immunity1999 67(8): 3998-4007) is also highly conserved, with 100% sequenceidentity across many strains. Full length Rv0125 includes an N-terminalsignal sequence which is cleaved to provide the mature protein.

In one embodiment the antigen is derived from Rv1196, such as comprise,such as consist of, a sequence having at least 70% identity to SEQ IDNo: 2, such as at least 80%, in particular at least 90%, especially atleast 95%, for example at least 98%, such as at least 99%. TypicalRv1196 related antigens will comprise (such as consist of) a derivativeof SEQ ID No: 2 having a small number of deletions, insertions and/orsubstitutions. Examples are those having deletions of up to 5 residuesat 0-5 locations, insertions of up to 5 residues at 0-5 five locationsand substitution of up to 20 residues. Other derivatives of Rv1196 arethose comprising (such as consisting of) a fragment of SEQ ID No: 2which is at least 200 amino acids in length, such as at least 250 aminoacids in length, in particular at least 300 amino acids in length,especially at least 350 amino acids in length.

In one embodiment the antigen is derived from Rv0125, such as comprise,such as consist of, a sequence having at least 70% identity to SEQ IDNo: 3, such as at least 80%, in particular at least 90%, especially atleast 95%, for example at least 98%, such as at least 99%. TypicalRv0125 related antigens will comprise (such as consist of) a derivativeof SEQ ID No: 3 having a small number of deletions, insertions and/orsubstitutions. Examples are those having deletions of up to 5 residuesat 0-5 locations, insertions of up to 5 residues at 0-5 five locationsand substitution of up to 20 residues. Other derivatives of Rv0125 arethose comprising (such as consisting of) a fragment of SEQ ID No: 3which is at least 150 amino acids in length, such as at least 200 aminoacids in length, in particular at least 250 amino acids in length,especially at least 300 amino acids in length. Particular derivatives ofRv0125 are those comprising (such as consisting of) the fragment of SEQID No: 3 corresponding to residues 1-195 of SEQ ID No: 3. Furtherimmunogenic derivatives of Rv0125 are those comprising (such asconsisting of) the fragment of SEQ ID No: 3 corresponding to residues192-323 of SEQ ID No: 3. Particularly preferred Rv0125 related antigensare derivatives of SEQ ID No: 3 wherein at least one (for example one,two or even all three) of the catalytic triad have been substituted ordeleted, such that the protease activity has been reduced and theprotein more easily produced—the catalytic serine residue may be deletedor substituted (e.g. substituted with alanine) and/or the catalytichistidine residue may be deleted or substituted and/or substituted thecatalytic aspartic acid residue may be deleted or substituted.Especially of interest are derivatives of SEQ ID No: 3 wherein thecatalytic serine residue has been substituted (e.g. substituted withalanine). Also of interest are Rv0125 related antigens which comprise,such as consist of, a sequence having at least 70% identity to SEQ IDNo: 3, such as at least 80%, in particular at least 90%, especially atleast 95%, for example at least 98%, such as at least 99% and wherein atleast one of the catalytic triad have been substituted or deleted orthose comprising, such as consisting of, a fragment of SEQ ID No: 3which is at least 150 amino acids in length, such as at least 200 aminoacids in length, in particular at least 250 amino acids in length,especially at least 300 amino acids in length and wherein at least oneof the catalytic triad have been substituted or deleted. Furtherimmunogenic derivatives of Rv0125 are those comprising (such asconsisting of) the fragment of SEQ ID No: 3 corresponding to residues192-323 of SEQ ID No: 3 wherein at least one (for example one, two oreven all three) of the catalytic triad have been substituted or deleted.Particular immunogenic derivatives of Rv0125 are those comprising (suchas consisting of) the fragment of SEQ ID No: 3 corresponding to residues1-195 of SEQ ID No: 3 wherein the catalytic serine residue (position 176of SEQ ID No: 3) has been substituted (e.g. substituted with alanine).

Suitably the antigen will comprise, such as consist of, a sequencehaving at least 70% identity to SEQ ID No. 4, such as at least 80%, inparticular at least 90%, especially at least 95%, such as at least 98%,for example at least 99%. Typical M72 related antigens will comprise,such as consist of, a derivative of SEQ ID No: 4 having a small numberof deletions, insertions and/or substitutions. Examples are those havingdeletions of up to 5 residues at 0-5 locations, insertions of up to 5residues at 0-5 five locations and substitution of up to 20 residues.Other derivatives of M72 are those comprising, such as consisting of, afragment of SEQ ID No: 4 which is at least 450 amino acids in length,such as at least 500 amino acids in length, such as at least 550 aminoacids in length, such as at least 600 amino acids in length, such as atleast 650 amino acids in length or at least 700 amino acids in length.As M72 is a fusion protein derived from the two individual antigensRv0125 and Rv1196, any fragment of at least 450 residues will comprise aplurality of epitopes from the full length sequence (Skeiky et al J.Immunol. 2004 172:7618-7628; Skeiky Infect. Immun. 1999 67(8):3998-4007;Dillon Infect. Immun. 1999 67(6):2941-2950).

M72 related antigen will comprise, such as consist of, a sequence havingat least 70% identity to SEQ ID No. 4, such as at least 80%, inparticular at least 90%, especially at least 95%, such as at least 98%,for example at least 99%.

Typical M72 related antigens will comprise, such as consist of, aderivative of SEQ ID No: 4 having a small number of deletions,insertions and/or substitutions. Examples are those having deletions ofup to 5 residues at 0-5 locations, insertions of up to 5 residues at 0-5five locations and substitution of up to 20 residues.

In particular embodiments the M72 related antigen will comprise residues2-723 of SEQ ID No. 4, for example comprise (or consist of) SEQ ID No. 4or comprise (or consist) of SEQ ID No. 5.

A further antigen that may be employed in accordance with the presentinvention is the tuberculosis antigen Rv1753 and variants thereof, suchas described in WO2010010180, for example a Rv1753 sequence selectedfrom Seq ID Nos: 1 and 2-7 of WO2010010180, in particular Seq ID No: 1.Another antigen of interest in the field of tuberculosis is Rv2386 andvariants thereof, such as described in WO2010010179, for example aRv2386 sequence selected from Seq ID Nos: 1 and 2-7 of WO2010010179, inparticular Seq ID No: 1. Other antigens of interest in the field oftuberculosis include Rv3616 and variants thereof, such as described inWO2011092253, for example a natural Rv3616 sequence selected from Seq IDNos: 1 and 2-7 of WO2011092253 or a modified Rv3616 sequence such asthose selected from Seq ID Nos: 161 to 169, 179 and 180 of WO2011092253,in particular Seq ID No: 167. An additional antigen of interest is HBHA,such as described in WO97044463, WO03044048 and WO2010149657. Theaforementioned patent applications WO2010010180, WO2010010179,WO2011092253, WO97044463, WO03044048 and WO2010149657 are incorporatedherein by reference in their entirety for the purpose of definingantigens which may be of use in the present invention.

Other antigens of interest are those comprising (or consisting of):Rv1174, also known as DPV, such as described in SEQ ID No 8 ofWO2010010177; Rv1793, also known as MTI or Mtb9.9, such as described inSEQ ID No 10 of WO2010010177; Rv2087, also known as MSL or Mtb9.8, suchas described in SEQ ID No 9 of WO2010010177; Rv3616, also known as HTCC1or Mtb40, such as described in SEQ ID Nos 1 and 2-7 WO2010010177 or SEQID Nos 161-169, 179 or 180 of WO2011092253; and/or Rv3874, also known asCFP10 or Tb38.1, such as described in SEQ ID No 9 of WO2010010177; or animmunogenic portion (such as at least 20, 50, 75 or 100 residuestherefrom) or variant thereof (such as having at least 70%, 80%, 90% or95% identity thereto). (WO2010010177 and WO2011092253 are incorporatedherein by reference in their entirety for the purpose of definingantigens which may be of use in the present invention).

Tuberculosis antigens are most suitably utilised in the form of apolypeptide, but may alternatively be provided in the form of apolynucleotide encoding said polypeptide. A further antigen that may beemployed in accordance with the present invention is derived fromVaricella zoster virus (VZV). The VZV antigen for use in the inventionmay be any suitable VZV antigen or immunogenic derivative thereof,suitably being a purified VZV antigen.

In one embodiment, the VZV antigen is the VZV glycoprotein gE (alsoknown as gp1) or immunogenic derivative hereof. The wild type or fulllength gE protein consists of 623 amino acids comprising a signalpeptide, the main part of the protein, a hydrophobic anchor region(residues 546-558) and a C-terminal tail. In one aspect, a gE C-terminaltruncate (also referred to truncated gE or gE truncate) is used wherebythe truncation removes 4 to 20 percent of the total amino acid residuesat the carboxy terminal end. In a further aspect, the truncated gE lacksthe carboxy terminal anchor region (suitably approximately amino acids547-623 of the wild type sequence). In a further aspect gE is atruncated gE having the sequence of SEQ ID NO. 6.The gE antigen, anchorless derivatives thereof (which are alsoimmunogenic derivatives) and production thereof is described inEP0405867 and references therein [see also Vafai A., Antibody bindingsites on truncated forms of varicalla-zoster virus gpl (gE)glycoprotein, Vaccine 1994 12:1265-9). EP192902 also describes gE andproduction thereof. Truncated gE is also described by Haumont et al.Virus Research (1996) vol 40, p199-204, herein incorporated fully byreference. An adjuvanted VZV gE composition suitable for use inaccordance of the present invention is described in WO2006/094756, i.e.a carboxyterminal truncated VZV gE in combination with adjuvantcomprising QS-21, 3D-MPL and liposomes further containing cholesterol.Leroux-Roels I. et al. (J. Infect. Dis. 2012, 206: 1280-1290) reportedon a phase I/Il clinical trial evaluating the adjuvanted VZV truncatedgE subunit vaccine.The antigen may comprise or consist of preparations derived from humanrespiratory syncytial virus (RSV). In certain favorable embodiments, apolypeptide antigen is an F protein polypeptide antigen from RSV.Particularly suitable as a polypeptide antigen component in the contextof the are conformationally constrained F polypeptide antigens.Conformationally constrained F proteins have previously been describedin both the prefusion (PreF) and postfusion (PostF) conformations. Suchconformationally constrained F proteins typically comprise an engineeredRSV F protein ectodomain. An F protein ectodomain polypeptide is aportion of the RSV F protein that includes all or a portion of theextracellular domain of the RSV F protein and lacks a functional (e.g.,by deletion or substitution) transmembrane domain, which can beexpressed, e.g., in soluble (not attached to a membrane) form in cellculture.Exemplary F protein antigens conformationally constrained in theprefusion conformation have been described in the art and are disclosedin detail in e.g., U.S. Pat. No. 8,563,002 (WO2009079796); US Publishedpatent application No. US201210093847 (WO2010/149745); US2011/0305727(WO2011/008974); US2014/0141037, WO2012/158613 and WO2014/160463 each ofwhich is incorporated herein by reference for the purpose ofillustrating prefusion F polypeptides (and nucleic acids), and methodsof their production. Typically, the antigen is in the form of a trimerof polypeptides. Additional publications providing examples of Fproteins in the prefusion conformation include: McLellan et al.,Science, Vol. 340: 1113-1117; McLellan et al., Science, Vol 342:592-598, and Rigter et al., PLOS One, Vol. 8: e71072, each of which canalso be used in the context of the immunogenic combinations disclosedherein.

For example, an F protein polypeptide stabilized in the prefusionconformation typically includes an ectodomain of an F protein (e.g., asoluble F protein polypeptide) comprising at least one modification thatstabilized the prefusion conformation of the F protein. For example, themodification can be selected from an addition of a trimerization domain(typically to the C terminal end), deletion of one or more of the furincleavage sites (at amino acids ˜105-109 and ˜133-136), a deletion of thepep27 domain, substitution or addition of a hydrophilic amino acid in ahydrophobic domain (e.g., HRA and/or HRB). In an embodiment, theconformationally constrained PreF antigen comprises an F2 domain (e.g.,amino acids 1-105) and an F1 domain (e.g., amino acids 137-516) of anRSV F protein polypeptide with no intervening furin cleavage sitewherein the polypeptide further comprises a heterologous trimerizationdomain positioned C-terminal to the F1 domain. Optionally, the PreFantigen also comprises a modification that alters glycosylation (e.g.,increases glycosylation), such as a substitution of one or more aminoacids at positions corresponding to amino acids ˜500-502 of an RSV Fprotein. When an oligomerization sequence is present, it is preferably atrimerization sequence. Suitable oligomerization sequences are wellknown in the art and include, for example, the coiled coil of the yeastGCN4 leucine zipper protein, trimerizing sequence from bacteriophage T4fibritin (“foldon”), and the trimer domain of influenza HA. Additionallyor alternatively, the F polypeptide conformationally constrained in theprefusion conformation can include at least two introduced cysteineresidues, which are in close proximity to one another and form adisulfide bond that stabilizes the pre-fusion RSV F polypeptide. Forexample, the two cysteines can be within about 10 Å of each other. Forexample, cysteines can be introduced at positions 165 and 296 or atpositions 155 and 290. An exemplary PreF antigen is represented by SEQID NO:7. The antigen may comprise or consist of preparations derivedfrom HIV. The antigen may be a HIV protein such as a HIV envelopeprotein. For example, the antigen may be a HIV envelope gp120polypeptide or an immunogenic fragment thereof.

One suitable antigen is the HIV clade B gp120 polypeptide of SEQ ID NO:8 of the published application WO 2008/107370 (or an immunogenicfragment of this polypeptide). SEQ ID NO: 8 of WO 2008/107370 isincorporated by reference into this application.

Suitable antigens also include a polypeptide comprising the V1V2 regionof SEQ ID NO: 1 of the published application WO 2015/036061, or animmunogenic derivative or fragment of the V1V2 region of SEQ ID NO: 1.In addition, a polypeptide comprising the V1V2 region of SEQ ID NO: 5 ofWO 2015/036061 or an immunogenic derivative or fragment of the V1V2region of SEQ ID NO: 5 may be used as a suitable antigen. SEQ ID NO: 1and SEQ ID NO: 5 of WO2015/036061 are incorporated by reference.

In another embodiment, the antigen may comprise two or more differentHIV envelope gp120 polypeptide antigens (or immunogenic fragments ofthese polypeptides). Suitable antigens include the and HIV clade C gp120polypeptide antigens including TV1 gp120 (SEQ ID No: 8) and 1086.C gp120(SEQ ID No: 9).

Other suitable HIV antigens include Nef, Gag and Pol HIV proteins andimmunogenic fragments thereof.

The composition may comprise non-typeable Haemophilus influenzaeantigen(s) for example selected from: Fimbrin protein [(U.S. Pat. No.5,766,608—Ohio State Research Foundation)] and fusions comprisingpeptides therefrom [e.g. LB1(f) peptide fusions; U.S. Pat. No. 5,843,464(OSU) or WO 99/64067]; OMP26 [WO 97/01638 (Cortecs)]; P6 [EP 281673(State University of New York)]; TbpA and/or TbpB; Hia; Hsf; Hin47; Hif;Hmw1; Hmw2; Hmw3; Hmw4; Hap; D15 (WO 94/12641); protein D (EP 594610);P2; and P5 (WO 94/26304); protein E (WO07/084053) and/or PiIA(WO05/063802). The composition may comprise Moraxella catarrhalisprotein antigen(s), for example selected from: OMP106 [WO 97/41731(Antex) & WO 96/34960 (PMC)]; OMP21; LbpA &/or LbpB [WO 98/55606 (PMC)];TbpA &/or TbpB [WO 97/13785 & WO 97/32980 (PMC)]; CopB [Helminen M E, etal. (1993) Infect. Immun. 61:2003-2010]; UspA1 and/or UspA2 [WO 93/03761(University of Texas)]; OmpCD; HasR (PCT/EP99/03824); PiIQ(PCT/EP99/03823); OMP85 (PCT/EP00/01468); lipo06 (GB 9917977.2); lipo10(GB 9918208.1); lipo11 (GB 9918302.2); lipo18 (GB 9918038.2); P6(PCT/EP99/03038); D15 (PCT/EP99/03822); OmplA1 (PCT/EP99/06781); Hly3(PCT/EP99/03257); and OmpE.

In an embodiment, the composition may comprise non-typeable H.influenzae (NTHi) protein antigen(s) and/or M. catarrhalis proteinantigen(s). The composition may comprise Protein D (PD) from H.influenzae. Protein D may be as described in WO91/18926. The compositionmay further comprise Protein E (PE) and/or Pilin A (PiIA) from H.Influenzae. Protein E and Pilin A may be as described in WO2012/139225.Protein E and Pilin A may be presented as a fusion protein; for exampleLVL735 as described in WO2012/139225. For example, the composition maycomprise three NTHi antigens (PD, PE and PiIA, with the two last onescombined as a PEPiIA fusion protein). The composition may furthercomprise UspA2 from M. catarrhalis. UspA2 may be as described inWO2015125118, for example MC-009 ((M)(UspA2 31-564)(HH)) described inWO2015125118. For example, the composition may comprise three NTHiantigens (PD, PE and PiIA, with the two last ones combined as a PEPiIAfusion protein) and one M. catarrhalis antigen (UspA2).

A plurality of antigens may be provided. For example, a plurality ofantigens may be provided to strengthen the elicited immune response(e.g. to ensure strong protection), a plurality of antigens may beprovided to broaden the immune response (e.g. to ensure protectionagainst a range of pathogen strains or in a large proportion of asubject population) or a plurality of antigens may be provided tocurrently elicit immune responses in respect of a number of disorders(thereby simplifying administration protocols). Where a plurality ofantigens are provided, these may be as distinct proteins or may be inthe form of one or more fusion proteins.

Antigen may be provided in an amount of 0.1 to 100 ug per human dose.

The present invention may be applied for use in the treatment orprophylaxis of a disease or disorder associated with one or moreantigens described above. In one embodiment the disease or disorder isselected from malaria, tuberculosis, COPD, HIV and herpes.

The adjuvant may be administered separately from an immunogen orantigen, or may be combined, either during manufacturing orextemporaneously), with an immunogen or antigen as an immunogeniccomposition for combined administration.

Sterilisation

For parenteral administration in particular, compositions should besterile. Sterilisation can be performed by various methods although isconveniently undertaken by filtration through a sterile grade filter.Sterilisation may be performed a number of times during preparation ofan adjuvant or immunogenic composition, but is typically performed atleast at the end of manufacture.

By “sterile grade filter” it is meant a filter that produces a sterileeffluent after being challenged by microorganisms at a challenge levelof greater than or equal to 1×10⁷/cm² of effective filtration area.Sterile grade filters are well known to the person skilled in the art ofthe invention for the purpose of the present invention, sterile gradefilters have a pore size between 0.15 and 0.25 um, suitably 0.18-0.22um, such as 0.2 or 0.22 um.

The membranes of the sterile grade filter can be made from any suitablematerial known to the skilled person, for example, but not limited tocellulose acetate, polyethersulfone (PES), polyvinylidene fluoride(PVDF), polytetrafluoroethylene (PTFE). In a particular embodiment ofthe invention one or more or all of the filter membranes of the presentinvention comprise polyethersulfone (PES), in particular hydrophilicpolyethersulfone. In a particular embodiment of the invention, thefilters used in the processes described herein are a double layerfilter, in particular a sterile filter with build-in prefilter havinglarger pore size than the pore size of the end filter. In one embodimentthe sterilizing filter is a double layer filter wherein the pre-filtermembrane layer has a pore size between 0.3 and 0.5 nm, such as 0.35 or0.45 nm. According to further embodiments, filters comprise asymmetricfilter membrane(s), such as asymmetric hydrophilic PES filtermembrane(s). Alternatively, the sterilizing filter layer may be made ofPVDF, e.g. in combination with an asymmetric hydrophilic PES pre-filtermembrane layer. In light of the intended medical uses, materials shouldbe of pharmaceutical grade (such as parenteral grade).

The teaching of all references in the present application, includingpatent applications and granted patents, are herein fully incorporatedby reference. A composition or method or process defined as “comprising”certain elements is understood to encompass a composition, method orprocess (respectively) consisting of those elements. As used herein,‘consisting essentially of’ means additional components may be presentprovided they do not alter the overall properties or function.

The invention will be further described by reference to the following,non-limiting, examples:

EXAMPLES Example 1—HPLC of a Crude Aqueous Extract of Quillaja saponariaMolina

Crude bark extract was separated by reverse phase HPLC using a C4 columnand gradient elution: mobile phase A—water/acetonitrile, 7/3 v/v with0.15% trifluoroacetic acid; mobile phase B—acetonitrile with 0.15%trifluoroacetic acid. UV detection was at 214 nm.

Crude bark extract samples are diluted as necessary with purified water.PVPP (60 mg/mL) was added, the mixture stirred for approximately 30minutes, and then centrifuged to separate the PVPP resin from thesupernatant.

The supernatant was then analysed to provide an HPLC UV chromatogram.

FIG. 1 provides a representative example of an HPLC UV chromatogram. Thepeak corresponding to the QS-21 fraction is indicated.

Example 2—Analytical Methods HPLC-UV Equipment

-   -   Waters Alliance 2690/2695 separations module    -   Waters 2487 UV Detector or 2996 PDA Detector    -   Vydac Protein C4 4.6×250 mm Sum column    -   Mobile Phase A (MPA)—0.15% trifluoroacetic acid in        water/acetonitrile (70:30 v/v)    -   Mobile Phase B (MPB)—0.15% trifluoroacetic acid in acetonitrile

Linear Gradient Conditions:

Flow rate Time (ml/min) % MPA % MPB 0 1 100 0 30 1 78.6 21.4 33 1 14.385.7

10 ul of sample is injected. UV detection is set at 214n M.

Using a blank injection for reference, integration of peaks in thechromatogram provides a total absorbance. Peak of interest (e.g. QS-21main peak) is compared to total absorbance to determine peak content asa percentage.

UPLC-UV Equipment

-   -   Waters Acquity UPLC    -   Waters Acquity Tunable UV Detector    -   Waters Acquity BEH C18 2.1×100 mm 1.7 um column    -   Mobile Phase A (MPA)—0.025% acetic acid in water/acetonitrile        (70:30 v/v)    -   Mobile Phase B (MPB)—0.025% trifluoroacetic acid in        water/acetonitrile (30:70 v/v)

Linear Gradient Conditions:

Flow rate Time (ml/min) % MPA % MPB 0 0.5 88 12 10.2 0.5 65.7 34.3 11.20.5 10 90 13.2 0.5 10 90

Column temperature 28 degrees C. 10 ul of sample is injected. UVdetection is set at 214 nM.

Using a blank injection for reference, integration of peaks in thechromatogram provides a total absorbance. Peak of interest (e.g. QS-21main peak) is compared to total absorbance to determine peak content asa percentage.

UPLC-UV/MS Equipment

-   -   Waters Acquity UPLC    -   Waters Acquity Tunable UV Detector    -   Waters Single-Quadrupole Mass Detector    -   Waters Acquity BEH C18 2.1×100 mm 1.7 um column    -   Mobile Phase A (MPA)—0.025% trifluoroacetic acid in        water/acetonitrile/isopropyl alcohol (75:20:5 v/v)    -   Mobile Phase B (MPB)—0.025% trifluoroacetic acid in        water/acetonitrile/isopropyl alcohol (10:72:18 v/v)

Linear Gradient Conditions:

Flow rate Time (ml/min) % MPA % MPB 0 0.6 100 0 6.23 0.6 23 77

Test sample is prepared in 0.2% acetic acid in water/acetonitrile (70:30v/v). Column temperature 55 degrees C. 10 ul of sample is injected. UVdetection is set at 214 nM.

Although retention times vary slightly between runs, the QS-21 group islocated at approximately 3.8 min (B-isomer) to approximately 4.5 minutes(prior to lyo impurity).

Using a blank injection for reference, integration of peaks in thechromatogram that elute after the solvent front between 0.5 and around5.50 minutes and do not appear in the blank is undertaken.

The monoisotope of the most abundant species is identified by combiningTIC over the entire chromatogram to create a combined spectrum.

Ratio of 2002 component to 1988 component is calculated by comparing theion current associated with the 2002 component with the ion currentassociated with the 1988 component within the QS-21 main peak.

FIG. 5 provides an exemplary chromatogram of a saponin extract accordingto the present invention. FIG. 6 shows expanded detail of the regionincluding the QS-21 group and lyo component.

FIG. 7 provides an exemplary extracted mass chromatograms for 1988 and2002 molecular weight ions of a purified Quillaja saponaria Molinasaponin extract.

Example 3—Purification of a Crude Aqueous Extract of Quillaja saponariaMolina

Crude aqueous extract of Quillaja saponaria Molina having a 2018component to QS-21 main peak ratio of 0.064 or lower and a Precedingpeak to QS-21 main peak ratio of 0.4 or lower, was treated with PVPP (1kg PVPP per litre of crude aqueous extract). After adsorption themixture was filtered to separate the PVPP and bound impurities from theliquor.

FIG. 2 provides an example HPLC-UV chromatogram for crude aqueousextract of Quillaja saponaria Molina (used for Preceding peak to QS-21main peak ratio determination).

FIG. 3 provides an example UPLC-UV chromatogram for crude aqueousextract of Quillaja saponaria Molina (used for 2018 component to QS-21main peak ratio determination).

Filtered liquor was concentrated and further purified byultrafiltration/diafiltration using a 30kD Hellicon membrane.

Resulting permeate was purified by reverse phase chromatography using apolystyrene resin (Amberchrom XT20) and the following conditions:

Initial Final Duration % % % % Step (min) Eluent A Eluent B Eluent AEluent B Injection + Rinse 11.3 100% 0% 100% 0% Gradient Elution 1 3.0100% 0%  71% 29%  Gradient Elution 2 50.0  71% 29%   53% 47%  GradientElution 3 3.0  53% 47%   0% 100%  Regeneration 10  0% 100%   0% 100% Gradient 3.0  0% 100%  100% 0% Equilibration 13.0 100% 0% 100% 0% EluentA: 5% Acetonitrile and 0.25% acetic acid Eluent B: 90% Acetonitrile and0.25% acetic acid Column: 30 cm ID Loading: 50-110 g per injection

Fractions were pooled to provide polystyrene purified saponin extractwith a composition:

-   -   % QS-21 main peak≥18% (by HPLC)    -   and    -   2018 component/QS-21 main peak ratio≤0.054 (by UPLC-UV).

FIG. 4 provides an example UPLC-UV chromatogram for a polystyrenepurified saponin extract pool.

The combined polystyrene purified fraction pool was further purified byreverse phase chromatography using a phenyl resin (EPDM) and thefollowing conditions:

Duration Step (min) % Eluent C % Eluent B Injection + Rinse 2.0 100% 0%Isocratic Elution 58.0 100% 0% Regeneration 5.0  0% 100%  Equilibration10.0 100% 0% Eluent B: 90% Acetonitrile and 0.25% acetic acid Eluent C:35.2% acetonitrile and 0.25% acetic acid Column: 45 cm ID Loading: 13-21g per injection

Fractions were pooled to provide phenyl purified saponin extract with acomposition:

-   -   % QS-21 group≥98.5    -   % QS-21 main peak≥94.5    -   %2018 component≤2.7%    -   Main peak outside of the QS-21 group≤1% (by UPLC-UV/MS).

The combined phenyl purified saponin extract was concentrated by captureand release with reverse phase chromatography using a C8 resin(Lichroprep RP8) and the following conditions: Loaded to columnconditioned at 24% acetonitrile and 0.20% acetic acid.

Eluted with 60% acetonitrile and 0.20% acetic acid.

-   -   11 cm column    -   Load: 50-142 g per injection

The C8 concentrated saponin extract was subjected to solvent exchangeusing ultrafiltration/diafiltration and a Pellicon 1 kDa membrane toreduce acetonitrile content below 21%.

The resulting solvent exchanged saponin extract was then lyophilised ina single step to provide the final product.

A series of process runs providing out of specification saponin extractwere found to arise from the use of crude bark extract with excessive2018 component content.

A plurality of runs according to the present invention, starting with acrude extract having a suitable 2018 component composition, wereperformed and on each occasion the final product was withinspecification.

The use of the process as described in Example 3 can consistentlyprovide a purified saponin extract of Quillaja saponaria Molina having adefined content in terms of QS-21 main peak and 2018 component, such asconsistently at least 93% of QS-21 main peak and 0.25-3% of 2018 andpresenting a chromatographic profile comparable to the chromatogramsshown in FIG. 5 , FIG. 6 and FIG. 7 .

Example 4—Immune Responses

6-8 week old female C57BL6 mice (5/group) were injected twiceintramuscularly with a 14-day interval with gE antigen formulated withthe adjuvant system AS01, a liposomal formulation comprising 3D-MPL andsaponin extract prepared according to the present intention. A controlgroup of 5 mice received gE with buffer alone.

Vaccine was prepared from three different lots of saponin extract andresults are provided for two different dosage levels (0.4 and 0.1 ug3D-MPL/QS-21 per animal corresponding to 1/125 and 1/500 of the humandose, respectively).

Spleens were collected at D21 and sera at D21 and D28, and analysed forT and B cell responses, respectively.

-   -   ICS (Intracellular Cytokine Staining)

Spleens were collected in RPMI medium and dissociated using a pottertissue grinder (homogenizer) using two up and down strokes. Homogenizedsamples were transferred to 50 ml polypropylene tubes. Fibrous materialwas removed by filtration through a 100 uM nylon cell strainer. Cellswere then washed, counted and re-suspended at 10⁷ cells per ml.

ICS is the technology which allows the quantification of antigenspecific T lymphocytes on the basis of cytokine production.

Lymphoid cells are re-stimulated overnight (O.N) in vitro with peptidesgE or medium in the presence of a protein transport inhibitor (brefeldinA). These cells are then processed by conventional immunofluorescentprocedure using fluorescent antibodies (extracellular staining: CD4,CD8; intracellular staining: TNF-alpha, IFN-gamma and IL2).

Results are expressed as a frequency of cytokine positive cells withinCD4 cell populations after subtraction of the medium condition for eachmouse. The data are presented for the population that showed expressionof at least two cytokines (IL2, IFN-alpha or TNF-alpha). Results areshown in FIG. 9 .

-   -   ELISA

Anti-gE total IgG were measured by ELISA. 96 well-plates were coatedwith antigen overnight at 4° C. The plates were then washed andsaturated with saturation buffer for 1 hour at 37° C. After, 100 ul ofdiluted mouse serum or standard or control was added and incubated for 1h 30 at 37° C. After wash, the plates were incubated for 1 hour at 37°C. with 100 ul anti mouse IgG-Biotinylated. After wash, the plates wereincubated for 30 min at 37° C. with 100 ul Streptavidin-POD conjugate.After wash, 100 ul of TMB per well was added and the plates were kept inthe dark at room temperature for 15 minutes. To stop the reaction, 100ul of H₂SO₄ 0.4N was added per well. The absorbance was read at awavelength of 450/630 nm by an Elisa plate reader. Results werecalculated using the Softmax-Pro software. Results are shown in FIG. 10(D21) and FIG. 11 (D28).

CONCLUSIONS

A consistent composition of saponin extract over different productionlots resulted in immunological responses with a limited degree ofvariation.

BIBLIOGRAPHY

-   Dalsgaard et al. in 1974 (“Saponin adjuvants”, Archiv. fur die    gesamte Virusforschung, Vol. 44, Springer Verlag, Berlin, p243-254)-   De Becker, G., V. Moulin, B. Pajak, C. Bruck, M. Francotte, C.    Thiriart, J. Urbain, and M. Moser. 2000. The adjuvant monophosphoryl    lipid A increases the function of antigen-presenting cells.    International immunology. 12:807-815.-   Didierlaurent A. M., Collignon C., Bourguignon P., Wouters S.,    Fierens K., Fochesato M., Dendouga N., Langlet C., Malissen B.,    Lambrecht B. N., Garcon N., Van Mechelen M., and S. Morel. 2014    Enhancement of Adaptive Immunity by the Human Vaccine Adjuvant AS01    Depends on Activated Dendritic Cells Journal of Immunology    193(4):1920-1930.-   Didierlaurent et al, 2017 Adjuvant system AS01: helping to overcome    the challenges of modern vaccines Expert Reviews of Vaccines 16(1):    55-63-   Garcon, N., and M. Van Mechelen. 2011. Recent clinical experience    with vaccines using MPL- and QS-21-containing adjuvant systems.    Expert review of vaccines. 10:471-486-   Ismaili, J., J. Rennesson, E. Aksoy, J. Vekemans, B. Vincart, Z.    Amraoui, F. Van Laethem, M. Goldman, and P. M. Dubois. 2002.    Monophosphoryl lipid A activates both human dendritic cells and T    cells. Journal of immunology. 168:926-932.-   Kensil, C. R., U. Patel, M. Lennick, and D. Marciani. 1991.    Separation and characterization of saponins with adjuvant activity    from Quillaja saponaria Molina cortex. Journal of immunology.    146:431-437.-   Kensil, C. R., and R. Kammer. 1998. QS-21: a water-soluble    triterpene glycoside adjuvant. Expert opinion on investigational    drugs. 7:1475-1482.-   Lambrecht, B. N., M. Kool, M. A. Willart, and H. Hammad. 2009.    Mechanism of action of clinically approved adjuvants. Current    opinion in immunology. 21:23-29.-   Leroux-Roels I. et al. J. Infect. Dis. 2012, 206: 1280-1290-   Li, H., S. B. Willingham, J. P. Ting, and F. Re. 2008. Cutting edge:    inflammasome activation by alum and alum's adjuvant effect are    mediated by NLRP3. Journal of immunology. 181:17-21.-   Livingston, P. O., S. Adluri, F. Helling, T. J. Yao, C. R.    Kensil, M. J. Newman, and D. Marciani. 1994. Phase 1 trial of    immunological adjuvant QS-21 with a GM2 ganglioside-keyhole limpet    haemocyanin conjugate vaccine in patients with malignant melanoma.    Vaccine. 12:1275-1280.-   Ragupathi, G., J. R. Gardner, P. O. Livingston, and D. Y. Gin. 2011.    Natural and synthetic saponin adjuvant QS-21 for vaccines against    cancer. Expert review of vaccines. 10:463-470-   Martin, M., S. M. Michalek, and J. Katz. 2003. Role of innate immune    factors in the adjuvant activity of monophosphoryl lipid A.    Infection and immunity. 71:2498-2507.-   Marty-Roix, R. et al. Identification of QS-21 as an    Inflammasome-activating Molecular Component of Saponin Adjuvants. J.    Biol. Chem. 291, 1123-36 (2016)-   Mata-Haro, V., C. Cekic, M. Martin, P. M. Chilton, C. R. Casella,    and T. C. Mitchell. 2007. The vaccine adjuvant monophosphoryl lipid    A as a TRIF-biased agonist of TLR4. Science. 316:1628-1632.-   Newman, M. J., J. Y. Wu, B. H. Gardner, K. J. Munroe, D.    Leombruno, J. Recchia, C. R. Kensil, and R. T. Coughlin. 1992.    Saponin adjuvant induction of ovalbumin-specific CD8+ cytotoxic T    lymphocyte responses. Journal of immunology. 148:2357-2362.-   Soltysik, S., J. Y. Wu, J. Recchia, D. A. Wheeler, M. J.    Newman, R. T. Coughlin, and C. R. Kensil. 1995. Structure/function    studies of QS-21 adjuvant: assessment of triterpene aldehyde and    glucuronic acid roles in adjuvant function. Vaccine. 13:1403-1410.

1-79. (canceled)
 80. An adjuvant composition comprising a liposomalformulation comprising a saponin extract; the saponin extract comprisingat least 93%, in sum and as determined by UV absorbance at 214 nm, ofone or more of:

and 0.25-3%, as determined by UV absorbance at 214 nm, of:


81. An adjuvant composition comprising a toll-like receptor-4 (TLR4)agonist and a saponin extract; the saponin extract comprising at least93%, in sum and as determined by UV absorbance at 214 nm, of one or moreof:

and 0.25-3%, as determined by UV absorbance at 214 nm, of:


82. The adjuvant composition according to claim 81, wherein the TLR4agonist is 3D-MPL.
 83. An immunogenic composition comprising: a) anadjuvant composition comprising a saponin extract comprising at least93%, in sum and as determined by UV absorbance at 214 nm, of one or moreof:

and 0.25-3%, as determined by UV absorbance at 214 nm, of:

and b) an immunogen or an antigen, or a polynucleotide encoding theimmunogen or the antigen.
 84. The immunogenic composition according toclaim 83 comprising the antigen; wherein the antigen is derived fromVaricella zoster virus.
 85. The immunogenic composition according toclaim 83 comprising the antigen; wherein the antigen is derived fromhuman respiratory syncytial virus.
 86. The adjuvant compositionaccording to claim 80, wherein the saponin extract comprises at least98%, in sum and as determined by UV absorbance at 214 nm, of one or moreof:

and 2118 component.
 87. The adjuvant composition according to claim 80,wherein the saponin extract comprises at least 65%, in sum and asdetermined by UV absorbance at 214 nm and by relative ion abundance, ofone or both of:


88. The adjuvant composition according to claim 80, wherein the saponinextract comprises at least 5% of:

by UV absorbance at 214 nm and by relative ion abundance.
 89. Theadjuvant composition according to claim 80, wherein the saponin extractcomprises at least 0.5% of:

by UV absorbance at 214 nm and by relative ion abundance.
 90. Theadjuvant composition according to claim 80, wherein the saponin extractcomprises at least 1% of:

by UV absorbance at 214 nm.
 91. The adjuvant composition according toclaim 81, wherein the saponin extract comprises at least 98%, in sum andby UV absorbance at 214 nm, of one or more of:

and 2118 component.
 92. The adjuvant composition according to claim 81,wherein the saponin extract comprises at least 65%, in sum and by UVabsorbance at 214 nm and by relative ion abundance, of one or both of:


93. The adjuvant composition according to claim 81, wherein the saponinextract comprises at least 5% of:

by UV absorbance at 214 nm and by relative ion abundance.
 94. Theadjuvant composition according to claim 81, wherein the saponin extractcomprises at least 0.5% of:

by UV absorbance at 214 nm and by relative ion abundance.
 95. Theadjuvant composition according to claim 81, wherein the saponin extractcomprises at least 1% of:

by UV absorbance at 214 nm.
 96. The immunogenic composition according toclaim 83, wherein the saponin extract comprises at least 98%, in sum andby UV absorbance at 214 nm, of one or more of:

and 2118 component.
 97. The immunogenic composition according to claim83, wherein the saponin extract comprises at least 65%, in sum and by UVabsorbance at 214 nm and by relative ion abundance, of one or both of:


98. The immunogenic composition according to claim 83, wherein thesaponin extract comprises at least 5% of:

by UV absorbance at 214 nm and by relative ion abundance.
 99. Theimmunogenic composition according to claim 83, wherein the saponinextract comprises at least 0.5% of:

by UV absorbance at 214 nm and by relative ion abundance.
 100. Theimmunogenic composition according to claim 83, wherein the saponinextract comprises at least 1% of:

by UV absorbance at 214 nm.